Immune checkpoint inhibition (ICI) is a promising cancer therapy, which has progressed rapidly from a preclinical concept to clinical implementation. Commonly considered targets in ICI are CTLA-4, PD-1/PD-L1, and LAG-3, and the list grows. As ICI is generally only beneficial for a subset of patients, there is a need to select patients that are eligible for therapy as well as to monitor therapy response. There is growing interest to do this noninvasively, by molecular imaging with target-specific tracers. To this day, noninvasive imaging has focused on CTLA-4 and PD-1/PD-L1, while there is no noninvasive tool available to accurately assess LAG-3 expression in vivo. In this proof-of-concept study, we developed nanobodies, the smallest functional fragments from camelid heavy chain-only antibodies, to noninvasively evaluate mouse LAG-3 expression using single positron emission computed tomography (SPECT)/CT imaging. The in vitro characterization of 114 nanobodies led to the selection of nine nanobodies binding to mouse LAG-3. The injection of ^99mTechnetium-labeled nanobodies in healthy mice showed specific uptake in immune peripheral organs like the spleen and lymph nodes, which was not observed in LAG-3 gene knock-out mice. Moreover, nanobody uptake could be visualized using SPECT/CT and correlated to the presence of LAG-3 as assessed in flow cytometry and immunohistochemistry. SPECT/CT scans of tumor bearing mice further confirmed the diagnostic potential of the nanobodies. These findings substantiate the approach to use nanobodies as a tool to image inhibitory immune checkpoints in the tumor environment.

Current cancer therapeutics suffer from a lack of specificity in targeting tumor cells and cause severe side effects. Therefore, the design of highly specialized drugs comprising antibody derivatives inducing apoptosis in targeted cancer cells is considered to be a promising strategy. Drugs acting on death receptor 5 (DR5) such as DR5 agonist antibodies replacing “TNF-related apoptosis-inducing ligand” (TRAIL) offer feasible opportunities in this direction. Although such agonists provided good antitumor activity in preclinical studies, they were less effective in clinical studies, possibly due to a disturbed Fc interaction with Fc-γ receptors. Thus, multimerized antigen binding fragments without Fc have been proposed to increase their efficacy. We generated nanobodies (Nbs), recombinant variable domains of heavy chain-only antibodies of camelids, against the DR5 ectodomain. Nb24 and Nb28 had an affinity in the nM and sub-nM range, but only Nb28 competes with TRAIL for binding to DR5. Bivalent, trivalent, and tetravalent constructs were generated, as well as an innovative pentameric Nb complex, to provoke avidity effects. In our cellular assays, these trimeric, tetrameric, and pentameric Nbs have a higher apoptotic capacity than monomeric Nbs and seem to mimic the activity of the natural TRAIL ligand on various cancer cells.